Parapoxvirus vectors

ABSTRACT

The present invention relates to recombinant  parapoxviruses  which carry in their genomes comprising heterologous DNA derived from a rabies virus, to the preparation of such constructs, and to their use in immunogenic compositions and vaccines. It further relates to the use of recombinant  parapoxviruses  for diagnostics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority, under 35 U.S.C. §119(e), to U.S.Provisional Application Ser. Nos. 61/346,988 and 61/414,287, filed onMay 21, 2010 and Nov. 16, 2010, respectively, the entire disclosures ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to recombinant parapoxviruses that containheterologous DNA derived from a rabies virus (RV) and to their use inimmunogenic compositions and vaccines. It also relates to methods forvaccinating against, treating, or preventing disease caused by rabiesvirus. It further relates to the use of recombinant parapoxviruses fordiagnostics.

BACKGROUND OF THE INVENTION

Viruses of the Poxyiridae family are oval, quite large, double-strandedDNA viruses. The genus Parapoxvirus (PPV) is included among theseviruses. They measure about 220-300 nm long by 140-170 nm wide. Theypossess a unique spiral coat that distinguishes them from the otherpoxviruses.

The PPV are divided into three different species. However, it has stillnot been clarified whether these viruses are autonomous species withinthe parapoxvirus genus or whether they are the same species. The firstspecies, Parapoxvirus ovis, is regarded as the prototype of the genus.It is also called ecthyma contagiosum virus, contagious pustulardermatitis virus, or orf virus. The second, Parapoxvirus bovis 1, isalso called bovine papular stomatitis virus or stomatitis papulosavirus. The third, Parapoxvirus bovis 2, is also called udderpoxvirus,paravaccinia virus, pseudocowpox virus, or milker's nodule virus.

Parapoxvirus species are endemic in ruminants. PPVs have been found inred deer, reindeer, red squirrels, and harbor seals. Infections with PPVcan cause local diseases in both animals and man. The zoonotic hosts ofPPV species are sheep, goats, and cattle. They cause infections inhumans through direct contact with infected animals, reacting withlocalized epidermal lesions which heal without scaring. Prophylacticmeasures, such as vaccines, can be used to control the diseases.

Vectors for expressing foreign genetic information based an avipox,racoonpox, capripox, swinepox, or vaccinia virus have been describedpreviously (see U.S. Pat. No. 5,942,235 and U.S. Pat. No. 7,094,412).Parapoxviruses represent different candidates that can be used in vectorvaccines. However, because of morphological, structural, and geneticdifferences between the individual genera of the poxviruses, the methodsused for these pox viruses cannot be used for Parapoxvirus. An exampleof such differences is that ORFV is missing a thymidine kinase (TK)gene, which is used for selection of recombinants in the differentorthopoxviruses. Also, some poxviruses have the ability to agglutinateerythrocytes, which is mediated by way of a surface protein, thehaemagglutinin, while Parapoxviruses do not.

PPV can have an immunomodulatory effect because they stimulategeneralized (non-specific) immune reactions in vertebrates. They havebeen used successfully in veterinary medicine for increasing generalresistance in animals. They can be combined with a homologous and/orheterologous antigen to provide vaccines that have a pathogen-specificeffect which lasts for months to years, as well as a rapidnon-pathogen-specific effect.

Parapoxvirus ovis has been used previously as a vector, as described inU.S. Pat. No. 6,365,393 and Rziha et al., 2000, J. Biotechnol, 83,137-145. It offers remarkable advantages when used as a vector,including a very narrow host range, lack of systemic infection,short-term vector-specific immunity (allowing repeated immunizations),early vaccination (induction of immunity can be started in presence ofmaternal antibodies), and beneficial immune modulating properties. Thepresent invention relates to using Parapoxvirus as a vector forheterologous DNA derived from rabies virus.

Parapoxvirus ovis strain D1701 is a highly attenuated strain that can bepropagated in cell culture with titres comparable to those of the wildtype virus. It has outstanding immune stimulating properties both inhosts that support replication of the infectious vector virus (e.g.,sheep and goats) and in hosts that do not (e.g., dogs, swine, horse,mouse, and rat). Zylexis®, formerly known as Baypamune®, which is apreparation of chemically inactivated Parapoxvirus ovis, derived fromstrain D1701, is used for the prophylaxis, metaphylaxis and therapeutictreatment of infectious diseases and for preventing stress-induceddiseases in animals.

The rabies virus (RV), Neurotropic lyssavirus, is a member of theRhabdoviridae family. It is a neurotrophic virus that causes fataldisease in humans and other mammals. Rabies is most often transmittedthrough the bite of a rabid animal, with transmission occurring throughthe saliva of the animals. The vast majority of rabies cases reported tothe United States Centers for Disease Control and Prevention (CDC) eachyear occur in wild animals like raccoons, skunks, bats, and foxes.Rabies is still a highly prevalent disease, especially in developingcountries. Approximately 50,000 people die each year from rabies. Thehighest risk of infection for humans is from rabid dogs.

The rabies virus is a single-stranded, negative-sense RNA virus whichencodes 5 proteins: nucleoprotein (N), phosphoprotein (P), matrixprotein (M), glycoprotein (G), and polymerase (L). The maturebullet-shaped virus, which averages approximately 180 nm in length and75 nm in width, has a ribonucleoprotein core, a protein coat, and alipid envelope. Glycoprotein projections, which are about 5-10 nm longand about 3 nm in diameter, cover the outer surface of the virus. Theyform approximately 400 trimeric tightly arranged spikes.

RV has a high affinity for nerve tissue. The reason for this is notknown. However, it may be that the rabies virus G protein can bind tothe acetylcholine receptor (a neurotransmitter receptor). After the RVattaches to the host cell via the viral G protein, the virus is absorbedinto the cell by engulfment.

SUMMARY OF THE INVENTION

The present invention generally relates to recombinant Parapoxviruses,and in particular Parapoxvirus ovis (PPVO). The recombinant parapoxvirusis used for mediating a rapid innate immune response, as well as along-lasting foreign gene-specific immunity against rabies virus. In oneembodiment, a recombinant parapoxvirus comprises heterologous DNAderived from a rabies virus. In one embodiment, the recombinantparapoxvirus comprises Parapoxvirus ovis strain D1701. In oneembodiment, the recombinant parapoxvirus comprises Parapoxvirus ovisstrain D1701-V.

In one embodiment, the recombinant parapoxvirus comprises the geneencoding the G protein of the rabies virus, or fragments thereof. In oneembodiment, the recombinant parapoxvirus comprises SEQ ID NO: 4 or apolynucleotide molecule having at least 98% identity to SEQ ID NO: 4. Inone embodiment, the heterologous DNA is inserted within the HindIIIfragment H/H of Parapoxvirus ovis strain D1701. In another embodiment,the heterologous DNA is inserted within the VEGF coding sequence oradjacent non-coding sequences within the HindIII fragment H/H ofParapoxvirus ovis strain D1701. In yet another embodiment, therecombinant parapoxvirus is Parapoxvirus ovis D1701-V-RabG.

The present invention embraces methods of preparing a recombinantparapoxvirus comprising inserting heterologous DNA into the genome ofthe parapoxvirus. In one embodiment, the method comprises the use ofParapoxvirus ovis. In one embodiment, the method comprises the use ofParapoxvirus ovis strain D1701. In one embodiment, the method comprisesthe use of Parapoxvirus ovis strain D1701-V. In one embodiment, theheterologous DNA used in the method comprises the gene encoding the Gprotein of the rabies virus, or fragments thereof. In one embodiment,the heterologous DNA used in the method comprises SEQ ID NO: 4 or apolynucleotide molecule having at least 98% identity to SEQ ID NO: 4. Inone embodiment of the method, the heterologous DNA is inserted withinthe HindIII fragment H/H of Parapoxvirus ovis strain D1701. In anotherembodiment, the heterologous DNA is inserted within the VEGF codingsequence or adjacent non-coding sequences within the HindIII fragmentH/H of Parapoxvirus ovis strain D1701. In one embodiment, the methodcomprises the preparation of Parapoxvirus ovis D1701-V-RabG. The presentinvention embraces an immunogenic composition comprising a recombinantparapoxvirus comprising heterologous DNA derived from a rabies virus anda carrier. The present invention embraces methods of preparing animmunogenic composition comprising combining a recombinant parapoxviruscomprising heterologous DNA derived from a rabies virus and a carrier.The present invention also embraces a vaccine comprising a recombinantparapoxvirus comprising heterologous DNA derived from a rabies virus anda carrier. The present invention embraces a method of preparing avaccine comprising combining a recombinant parapoxvirus comprisingheterologous DNA derived from a rabies virus and a carrier. In some ofthese embodiments, the recombinant parapoxvirus comprises Parapoxvirusovis, while in some the recombinant parapoxvirus comprises Parapoxvirusovis strain D1701, and in others the recombinant parapoxvirus comprisesParapoxvirus ovis strain D1701-V. In some of these embodiments, theheterologous DNA comprises the gene encoding the G protein of the rabiesvirus, or fragments thereof, while in other embodiments, theheterologous DNA comprises SEQ ID NO: 4 or a polynucleotide moleculehaving at least 98% identity to SEQ ID NO: 4. In some of theseembodiments, the heterologous DNA is inserted within the HindIIIfragment H/H of Parapoxvirus ovis strain D1701, in other of theseembodiments, the heterologous DNA is inserted within the VEGF codingsequence or adjacent non-coding sequences within the HindIII fragmentH/H of Parapoxvirus ovis strain D1701. In some of these embodiments, therecombinant parapoxvirus is Parapoxvirus ovis D1701-V-RabG.

The present invention embraces a method of inducing in an animal animmune response against rabies virus, comprising administering to saidanimal an immunologically effective amount of an immunogenic compositioncomprising a recombinant parapoxvirus comprising heterologous DNAderived from a rabies virus and a carrier. The present inventionembraces a method of vaccinating an animal against rabies virus,comprising administering to said animal a therapeutically effectiveamount of a vaccine composition comprising the recombinant parapoxviruscomprising heterologous DNA derived from a rabies virus and a carrier.The present invention embraces a use of a recombinant parapoxviruscomprising heterologous DNA derived from a rabies virus in thepreparation of a medicament for inducing an immune response againstrabies virus in an animal. The present invention embraces a use of arecombinant parapoxvirus comprising heterologous DNA derived from arabies virus in the preparation of a medicament for vaccinating ananimal against rabies virus. In some of these embodiments, therecombinant parapoxvirus comprises Parapoxvirus ovis, while in some therecombinant parapoxvirus comprises Parapoxvirus ovis strain D1701, andin others the recombinant parapoxvirus comprises Parapoxvirus ovisstrain D1701-V. In some of these embodiments, the heterologous DNAcomprises the gene encoding the G protein of the rabies virus, orfragments thereof, while in other embodiments, the heterologous DNAcomprises SEQ ID NO: 4 or a polynucleotide molecule having at least 98%identity to SEQ ID NO: 4. In some of these embodiments, the heterologousDNA is inserted within the HindIII fragment H/H of Parapoxvirus ovisstrain D1701. In other of these embodiments, the heterologous DNA isinserted within the VEGF coding sequence or adjacent non-codingsequences within the HindIII fragment H/H of Parapoxvirus ovis strainD1701. In some of these embodiments, the recombinant parapoxvirus isParapoxvirus ovis D1701-V-RabG. In some of these embodiments, an anti-Gprotein-specific protective immune response is induced. In other suchembodiments, the immune response is the induction of anti-G proteinserum antibodies. In yet other such embodiments, the induction resultsin antibody titers exceeding 0.5 International Units per ml.

The present invention provides methods of determining the origin of aParapoxvirus present in an animal. The Parapoxviruses described hereincan be distinguished from wild-type strains in both their genomiccomposition and proteins expressed. Such distinction allows fordiscrimination between vaccinated and infected animals. The presentinvention encompasses a use of a recombinant parapoxvirus as taughtherein in an assay for the differentiation of infected from vaccinatedanimals (DIVA). In one embodiment, the recombinant Parapoxvirus ovisD1701-V-RabG is used in a DIVA assay.

These and other embodiments are disclosed and encompassed by thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the present invention will be gained byreferring to the accompanying drawings in which:

FIG. 1: Construction of plasmid pdV-RabG. The G gene of rabies virus isinserted as a BamHI-EcoRI fragment into the multiple cloning site (boxedbases) of pdV-Rec1, which resulted in plasmid pdV-RabG (7.7 kbp insize). The position of primers ORF32N (SEQ ID NO: 1) and ORF31N (SEQ IDNO: 2) are shown. SEQ ID NO: 3 is shown in the Figure.

Abbreviations: Sm=SmaI, H3=HindIII, EcoV=EcoRV, P=PstI, B=BamHI, K=KpnI,E=EcoRI, S=SalI. F9L and ORF3 indicate the presence of the PPVO genesF9L (ORF 131) and ORF3 (ORF 133). Pvegf indicates the presence of thevegF-E promoter used to control expression of the inserted G gene. (P)and (H3) depict the destroyed PstI- and HindIII-restrictions sites ofthe vector backbone of plasmid pSPT-18, the T7 and SP6 promoters ofpSPT-18 are indicated, respectively. The figure is not drawn to scale.

FIG. 2: Virus neutralizing serum antibody (SNT) response afterimmunization with different doses of D1701-V-RabG. Groups of C57/BL6mice (for each day n=6 or 7) were immunized with a single dose ofD1701-V-RabG using 10⁷ PFU (plaque-forming units) (A), 10⁶ PFU (B), 10⁵PFU (C), and 10⁴ PFU (D). Individual sera were taken daily during 14days after immunization (d1 to d14).

FIG. 3: Challenge experiment of immunized mice, C57/BL6 mice received asingle dose of D1701-V-RabG as indicated (10⁷ to 10⁴ PFU) and wereintracranially challenge infected 2 weeks later with at least 3,400 LD₅₀(4.8×10⁵ PFU) of RV virulent strain CVS. (A) shows the survival curvesof the individual animals in each group, whereas in (B) the same resultswere plotted in percentage of survivors.

FIG. 4. SEQ ID NO: 4—Sequence of the full length coding region of the Ggene of the rabies virus (1575 nt) plus 7 nt on the 5′-end and 6 nt onthe 3′-end as linker sequences for restriction enzyme analysis (BamHIand EcoRI).

FIG. 5. Role of T-cells for protective immunity. CD4-Imm is the groupimmunized with antisera specific for CD4 cells. CD8-Imm is the groupimmunized with antisera specific for CD8-T-cells. CD4/8-Imm is the groupimmunized with antisera specific for CD4/CD8-T-cells. Imm C is thecontrol group vaccinated with D1701-V-RabG, which did not receiveantisera. Non-Imm C is the control group that was not vaccinated withD1701-V-RabG and did not receive antisera. “Days p. chall” is the numberof days post challenge with the rabies strain CVS.

FIG. 6. Protection seen with post-exposure vaccination. Non-Imm is thecontrol group that was not vaccinated with D1701-V-RabG. D1701-V-RabG isthe group that was vaccinated with D1701-V-RabG. CVS is the virulentrabies virus strain. “Days p. chall” is the number of days postchallenge with the RV strain CVS.

FIG. 7. Protection seen with various post-exposure vaccination regimens.Gray squares indicate the days on which mice were vaccinated withD1701-V-RabG.

FIG. 8. Serum antibody response (determined as serum neutralizationantibodies, SNT) 6 days and 13 days after immunization. Mice wereimmunized with D1701-V-RabG intravaginally (i.vag.), by scarification,intraperitoneally (i.p.), intradermally (i.d.), intransally (i.n.),subcutaneously (s.c.), intramuscularly (i.m.), or intraveneously (i.v.).

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

The following definitions may be applied to terms employed in thedescription of embodiments of the invention. They supersede anycontradictory definitions contained in each individual referenceincorporated herein by reference.

“About” or “approximately,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value (e.g., within the 95% confidence interval for the mean)or within 10 percent of the indicated value, whichever is greater,unless about is used in reference to time intervals in weeks where“about 3 weeks,” is 17 to 25 days, and about 2 to about 4 weeks is 10 to40 days.

“Adjuvant”, as used herein, refers to any substance which serves as anon-specific stimulator of the immune response. See below for a furtherdescription of adjuvants.

The term “animal” or “animal subject”, as used herein, includes anyanimal that is susceptible to rabies infections, including mammals, bothdomesticated and wild.

“Antibody”, as used herein, is any polypeptide comprising anantigen-binding site regardless of the source, method of production, orother characteristics. It refers to an immunoglobulin molecule or afragment thereof that specifically binds to an antigen as the result ofan immune response to that antigen. Immunoglobulins are serum proteinscomposed of “light” and “heavy” polypeptide chains having “constant” and“variable” regions and are divided into classes (e.g., IgA, IgD, IgE,IgG, and IgM) based on the composition of the constant regions. Anantibody that is “specific” for a given antigen indicates that thevariable regions of the antibody recognize and bind a specific antigenexclusively. Antibodies can be a polyclonal mixture or monoclonal.Antibodies can be intact immunoglobulins derived from natural sources orfrom recombinant sources, or can be immunoreactive portions of intactimmunoglobulins. An “antibody” can be converted to an antigen-bindingprotein, which includes but is not limited to antibody fragments.

The term “antigen” or “immunogen”, as used herein, refers to a moleculethat contains one or more epitopes (linear, conformational or both) thatupon exposure to a subject will induce an immune response that isspecific for that antigen. The term “antigen” can refer to attenuated,inactivated or modified live bacteria, viruses, fungi, parasites orother microbes. The term “antigen” as used herein can also refer to asubunit antigen, which is separate and discrete from a whole organismwith which the antigen is associated in nature. The term antigen alsorefers to antibodies, such as anti-idiotype antibodies or fragmentsthereof, and to synthetic peptide mimotopes that can mimic an antigen orantigenic determinant (epitope). The term “antigen” can also refer to anoligonucleotide or polynucleotide that expresses an antigen or antigenicdeterminant in vivo, such as in DNA immunization applications.

“Buffer” means a chemical system that prevents change in theconcentration of another chemical substance, e.g., proton donor andacceptor systems serve as buffers preventing marked changes in hydrogenion concentration (pH). A further example of a buffer is a solutioncontaining a mixture of a weak acid and its salt (conjugate base) or aweak base and its salt (conjugate acid).

The term “cell line” or “host cell”, as used herein, means a prokaryoticor eukaryotic cell in which a virus can replicate or be maintained.

“Cellular immune response” or “cell mediated immune response” is onemediated by T-lymphocytes or other white blood cells or both, andincludes the production of cytokines, chemokines and similar moleculesproduced by activated T-cells, white blood cells, or both.

“Conservative substitution” is defined in the art and known to oneskilled in the art, and is recognized to classify residues according totheir related physical properties.

The term “culture”, as used herein, means a population of cells ormicroorganisms growing in the absence of other species or types.

The term “DIVA” as used herein means to Differentiate Infected fromVaccinated Animals.

“Dose” refers to a vaccine or immunogenic composition given to asubject. A “first dose” or “priming vaccine” refers to the dose of sucha composition given on Day 0. A “second dose” or a “third dose” or an“annual dose” refers to an amount of such composition given subsequentto the first dose, which may or may not be the same vaccine orimmunogenic composition as the first dose.

An “epitope” is the specific site of the antigen which binds to a T-cellreceptor or specific antibody, and typically comprises from about 3amino acid residues to about 20 amino acid residues.

“Excipient” refers to any component of a vaccine or immunogeniccomposition that is not an antigen.

“Fragment” refers to a truncated portion of a protein or gene.“Functional fragment” and “biologically active fragment” refer to afragment that retains the biological properties of the full lengthprotein or gene. An “immunogenically active fragment” refers to afragment that elicits an immune response.

The term “G protein”, as used herein, refers to protein in theglycoprotein projections that cover the outer surface of a rabies virus.

The term “heterologous”, as used herein, means derived from a differentspecies or strain.

The term “homologous”, as used herein, means derived from the samespecies or strain.

“Homology” or “percent homology” refers to the percentage of nucleotideor amino acid residues in the candidate sequence that are identical withthe residues in the comparator sequences after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and also considering any conservative substitutionsas part of the sequence identity.

“Humoral immune response” refers to one that is at least in partmediated by antibodies.

“Identity” or “percent identity” refers to the percentage of nucleotidesor amino acids in the candidate sequence that are identical with theresidues in the comparator sequence after aligning both sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity.

“Immune response” in a subject refers to the development of a humoralimmune response, a cellular immune response, or a humoral and a cellularimmune response to an antigen. The immunogenic response may besufficient for diagnostic purposes or other testing, or may be adequateto prevent signs or symptoms of disease, including adverse healtheffects or complications thereof, caused by infection with a diseaseagent. Immune responses can usually be determined using standardimmunoassays and neutralization assays, which are known in the art.

“Immunogenic” or “Immunogenicity”, as used herein, refers to thecapability to elicit an immune response directed specifically against anantigen.

The terms “immunogenic composition,” or “immunologically effectiveamount,” or “amount effective to produce an immune response,” as usedherein, refer to a composition or antigen capable of being recognized bythe immune system, resulting in the generation of a specific immuneresponse (i.e., has immunogenic activity) when administered alone orwith a pharmaceutically acceptable carrier, to an animal.

“Isolated”, as used herein, means removed from its naturally occurringenvironment, either alone or in a heterologous host cell, or chromosomeor vector (e.g., plasmid, phage, etc.). “Isolated bacteria,” “isolatedanaerobic bacteria,” “isolated bacterial strain,” “isolated virus”“isolated viral strain” and the like refer to a composition in which thebacteria or virus are substantial free of other microorganisms, e.g., ina culture, such as when separated from it naturally occurringenvironment. “Isolated,” when used to describe any particularly definedsubstance, such as a polynucleotide or a polypeptide, refers to thesubstance that is separate from the original cellular environment inwhich the substance—such as a polypeptide or nucleic acid—is normallyfound. As used herein therefore, by way of example only, a recombinantcell line constructed with a polynucleotide of the invention makes useof the “isolated” nucleic acid. Alternatively, if a particular proteinor a specific immunogenic fragment is claimed or used as a vaccine orother composition, it would be considered to be isolated because it hadbeen identified, separated and to some extent purified as compared tohow it may exist in nature. If the protein or a specific immunogenicfragment thereof is produced in a recombinant bacterium or eukaryoteexpression vector that produces the antigen, it is considered to existas an isolated protein or nucleic acid. For example, a recombinant cellline constructed with a polynucleotide makes use of an “isolated”nucleic acid.

“Medicinal agent” refers to any agent which is useful in the prevention,cure, or improvement of disease, or the prevention of some physiologicalcondition or occurrence.

The term “multiplicity of infection” (MOI) refers to a ratio of thenumber of organisms per cell, which details how much inoculum is goingto be used in a given infection.

The terms “parapoxvirus”, “parapoxvirus strains”, as used herein, referto viruses belonging to the family Poxyiridae and the genusParapoxvirus.

The terms “Parapoxvirus ovis” and “Parapoxvirus ORFV”, as used herein,refer to viruses belonging to the family Poxyiridae, the genusParapoxvirus, and the species Parapoxvirus ovis. These viruses are alsocalled ecthyma contagiosum virus, contagious pustular dermatitis virus,or orf virus. They possess a unique spiral coat that distinguishes themfrom the other poxviruses.

The term “Parapoxvirus ovis strain D1701” refers to the virus asdescribed in U.S. Pat. No. 6,365,393, which is incorporated herein byreference. “Parapoxvirus ovis strain D1701-V” refers to Parapoxvirusovis strain D1701 adapted to the simian cell line Vero.

“Parenteral administration” refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of a routethat does not include the digestive tract. Parenteral administrationincludes subcutaneous, intramuscular, transcutaneous, intradermal,intraperitoneal, intraocular, and intravenous administration.

The term “pathogen” or “pathogenic microorganism”, as used herein, meansa microorganism—for example a rabies virus—which is capable of inducingor causing a disease, illness, or abnormal state in its host animal.

“Pharmaceutically acceptable” refers to substances, which are within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit-to-riskratio, and effective for their intended use.

The term “poxvirus”, as used herein, refers to viruses belonging to thefamily Poxyiridae. These viruses are oval, quite large, double-strandedDNA viruses.

The term “polynucleotide” or “polynucleotide molecule”, as used herein,means an organic polymer molecule composed of nucleotide monomerscovalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA(ribonucleic acid) are examples of polynucleotides with distinctbiological function.

The terms “prevent”, “preventing” or “prevention”, and the like, as usedherein, mean to inhibit the replication of a microorganism, to inhibittransmission of a microorganism, or to inhibit a microorganism fromestablishing itself in its host. These terms and the like as used hereincan also mean to inhibit or block one or more signs or symptoms ofinfection. The treatment is considered therapeutic if there is areduction in the microorganism load.

“Protection”, “protecting”, and the like, as used herein with respect toa vaccine or other composition, means that the vaccine or compositionprevents or reduces the symptoms of the disease caused by the organismfrom which the antigen(s) used in the vaccine or composition is derived.The terms “protection” and “protecting” and the like, also mean that thevaccine or composition can be used to therapeutically treat the diseaseor one of more symptoms of the disease that already exists in a subject.

The term “rabies virus” refers to Neurotropic lyssavirus, a member ofthe Rhabdoviridae family. It is a single-stranded, negative-sense RNAvirus which has glycoprotein projections on its outer surface.

“Recombinant PPV” or “Recombinant PPV” are PPV having insertions and/ordeletions in their genome. The insertions and deletions are preparedusing molecular biological methods.

“Species homologs” include genes found in two or more different specieswhich possess substantial polynucleotide sequence homology and possessthe same, or similar, biological functions and/or properties. Preferablypolynucleotide sequences which represent species homologs will hybridizeunder moderately stringent conditions, as described herein by example,and possess the same or similar biological activities and or properties.In another aspect, polynucleotides representing species homologs willshare greater than about 60% sequence homology, greater than about 70%sequence homology, greater than about 80% sequence homology, greaterthan about 90% sequence homology, greater than about 95% sequencehomology, greater than about 96% sequence homology, greater than about97% sequence homology, greater than about 98% sequence homology, orgreater than about 99% sequence homology.

The terms “specific binding,” “specifically binds,” and the like, aredefined as two or more molecules that form a complex that is measurableunder physiologic or assay conditions and is selective. An antibody orother inhibitor is said to “specifically bind” to a protein if, underappropriately selected conditions, such binding is not substantiallyinhibited, while at the same time non-specific binding is inhibited.Specific binding is characterized by high affinity and is selective forthe compound or protein. Nonspecific binding usually has low affinity.Binding in IgG antibodies, for example, is generally characterized by anaffinity of at least about 10⁻⁷ M or higher, such as at least about 10⁻⁸M or higher, or at least about 10⁻⁹ M or higher, or at least about 10⁻¹⁰or higher, or at least about 10⁻¹¹ M or higher, or at least about 10⁻¹²M or higher. The term is also applicable where, e.g., an antigen-bindingdomain is specific for a particular epitope that is not carried bynumerous antigens, in which case the antibody carrying theantigen-binding domain will generally not bind other antigens.

“Specific immunogenic fragment”, as used herein, refers to a portion ofa sequence that is recognizable by an antibody or T cell specific forthat sequence.

“Subject” refers to any animal that is susceptible to rabies infections,including mammals, both domesticated and wild.

“Substantially identical”, as used herein, refers to a degree ofsequence identity of at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about99%.

“Therapeutically effective amount”, as used herein, refers to an amountof an antigen or vaccine or composition that would induce an immuneresponse in a subject (e.g., dog) receiving the antigen or vaccine orcomposition which is adequate to prevent or ameliorate signs or symptomsof disease, including adverse health effects or complications thereof,caused by infection with a pathogen, such as a virus, bacterium,parasite or fungus. Humoral immunity or cell-mediated immunity, or bothhumoral and cell-mediated immunity, can be induced. The immunogenicresponse of an animal to an antigen, vaccine, or composition can beevaluated indirectly through measurement of antibody titers, lymphocyteproliferation assays, or directly through monitoring signs and symptomsafter challenge with the wild type strain. The protective immunityconferred by a vaccine or composition can be evaluated by measuringreduction of challenge organism shed, and/or reduction in clinicalsigns, such as mortality, morbidity, temperature, and overall physicalcondition, health, and performance of the subject. The amount of avaccine or composition that is therapeutically effective can vary,depending on the particular immunogen used, or the condition of thesubject, and can be determined by one skilled in the art.

The terms “treat”, “treating” or “treatment”, and the like, as usedherein, mean to reduce or eliminate an infection by a microorganism.These terms and the like can also mean to reduce the replication of amicroorganism, to reduce the transmission of a microorganism, or toreduce the ability of a microorganism to establish itself in its host.These terms and the like as used herein can also mean to reduce,ameliorate, or eliminate one or more signs or symptoms of infection by amicroorganism, or accelerate the recovery from infection by amicroorganism.

The terms “vaccinate” and “vaccinating” and the like, as used herein,mean to administer to an animal a vaccine or immunogenic composition.

The terms “vaccine” and “vaccine composition,” as used herein, mean acomposition which prevents or reduces an infection, or which prevents orreduces one or more signs or symptoms of infection. The protectiveeffects of a vaccine composition against a pathogen are normallyachieved by inducing in the subject an immune response. Generallyspeaking, abolished or reduced incidences of infection, amelioration ofthe signs or symptoms, or accelerated elimination of the microorganismfrom the infected subjects are indicative of the protective effects of avaccine composition. The vaccine compositions of the present inventionprovide protective effects against infections caused by rabies virus.

The term “variant,” as used herein, refers to a derivation of a givenprotein and/or gene sequence, wherein the derived sequence isessentially the same as the given sequence, but for mutationaldifferences. Said differences may be naturally-occurring, orsynthetically- or genetically-generated.

A “vector” or a “vector virus” is a PPV which is suitable for theinsertion of heterologous DNA, which can transport the inserted DNA intocells or organisms, and which, where appropriate, enables theheterologous DNA to be expressed.

The term “veterinarily acceptable carrier” as used herein refers tosubstances, which are within the scope of sound medical judgment,suitable for use in contact with the tissues of animals without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit-to-risk ratio, and effective for their intendeduse.

The following description is provided to aid those skilled in the art inpracticing the present invention. Even so, this description should notbe construed to unduly limit the present invention as modifications andvariations in the embodiments discussed herein can be made by those ofordinary skill in the art without departing from the spirit or scope ofthe present inventive discovery.

Viruses, Immunogenic Compositions, and Vaccines

The present invention embraces the use of parapoxviruses for thepreparation of a recombinant parapoxvirus comprising heterologous DNAderived from a rabies virus.

In one embodiment, Parapoxvirus ovis (PPVO) for the preparation of arecombinant parapoxvirus comprising heterologous DNA derived from arabies virus is used. In another embodiment, the Parapoxvirus ovisstrain D1701 is used. In a further embodiment, the Parapoxvirus ovisstrain D1701-V is used.

The genetic sequence inserted into the parapoxvirus includesheterologous DNA derived from a rabies virus. In one embodiment, theheterologous DNA comprises the gene encoding the G protein of the rabiesvirus, or fragments thereof. In one embodiment, the heterologous DNAcomprises SEQ ID NO: 4 or a polynucleotide molecule having at least 98%identity to SEQ ID NO: 4. The structure of this gene is furtherdisclosed, for example, by Anilionis et al., Nature 294, 275-278 (1981).The insert is 1588 nt in size. The complete sequence of the insert (SEQID NO: 4) is shown in FIG. 4. It contains the full-length coding regionof the 0 gene of rabies virus (1575 nt) plus 7 nt on the 5′-end and 6 nton the 3′-end as linker sequences for restriction enzyme analysis (BamHIand EcoRI).

Knowledge of the sequence of a polynucleotide makes readily availableevery possible fragment of that polynucleotide. The invention thereforeprovides for fragments of the G protein. In one embodiment, functionalfragments are provided for. In another embodiment, biologically activefragments are provided for. Fragments can be purified by conventionalmethods, such as for example by filtration or chromatography. Fragmentscan be produced by recombination by methods known to one skilled in thearts.

In preparing the recombinant parapoxvirus, the heterologous DNA isinserted within the HindIII fragment H/H of Parapoxvirus ovis strainD1701. In another embodiment, the heterologous DNA is inserted in withinthe VEGF coding sequence or adjacent non-coding sequences within theHindIII fragment H/H of Parapoxvirus ovis strain D1701. The methods usedto insert the heterologous DNA into the parapoxvirus are standard andknown to one skilled in the art. They are described in U.S. Pat. No.6,365,393.

In one embodiment, the recombinant parapoxvirus comprising heterologousDNA derived from a rabies virus is Parapoxvirus ovis D1701-V-RabG.

In one embodiment, the recombinant parapoxvirus comprising heterologousDNA derived from a rabies virus is D1701-VrV RabG (K-UC1002), which isdeposited at The American Type Culture Collection (ATCC®), Manassas,Va., 20108 USA with ATCC® Patent Deposit Designation PTA-11662, incompliance with Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure.

The sequence of the plasmid pdV-RabG (7.692 nt) is SEQ ID NO: 5, whichis shown in the sequence listing.

The invention also embraces polynucleotide sequences that have at leastabout 99%, at least about 98%, at least about 97%, at least about 96%,at least about 95%, at least about 93%, at least about 90%, at leastabout 85%, at least about 80%, at least about 75%, at least about 70%,at least about 65%, at least about 60%, at least about 55%, and at leastabout 50% identity and/or homology to the sequences described herein.

The invention also embraces polynucleotide sequences which hybridizeunder moderately to highly stringent conditions to the non-codingstrand, or complement, of any one of the SEQ ID NOs described herein,and species homologs thereof. Exemplary high stringency conditionsinclude a final wash in buffer comprising 0.2× SSC/0.1% SDS, at 65° C.to 75° C., while exemplary moderate stringency conditions include afinal wash in buffer comprising 2×SSC/0.1% SDS, at 35° C. to 45° C. Itis understood in the art that conditions of equivalent stringency can beachieved through variation of temperature and buffer, or saltconcentration as described in Ausubel, et al. (Eds.), Protocols inMolecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.

The recombinant PPV can be propagated in cells, cell lines and hostcells. Said cells, cell lines, or host cells may be for example, but arenot limited to, mammalian cells and non-mammalian cells. Cells, celllines, and host cells in which the PPV can be propagated are readilyknown and accessible to those of ordinary skill in the art. In oneembodiment Vero cells are used. In other embodiments, bovine kidney orovine testis cells are used.

The recombinant PPV can be further attenuated or inactivated prior touse in an immunogenic composition or vaccine. Methods of attenuation andinactivation are well known to those skilled in the art. Methods forattenuation include, but are not limited to, serial passage in cellculture on a suitable cell line, ultraviolet irradiation, and chemicalmutagenesis. Methods for inactivation include, but are not limited to,treatment with formalin, betapropriolactone (BPL) or binaryethyleneimine (BEI), or other methods known to those skilled in the art.

Inactivation by formalin can be performed by mixing the virus suspensionwith 37% formaldehyde to a final formaldehyde concentration of 0.05%.The virus-formaldehyde mixture is mixed by constant stirring forapproximately 24 hours at room temperature. The inactivated virusmixture is then tested for residual live virus by assaying for growth ona suitable cell line.

Inactivation by BEI can be performed by mixing the virus suspension ofthe present invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 NNaOH) to a final BEI concentration of 1 mM. The virus-BEI mixture ismixed by constant stirring for approximately 48 hours at roomtemperature, followed by the addition of 1.0 M sodium thiosulfate to afinal concentration of 0.1 mM. Mixing is continued for an additional twohours. The inactivated virus mixture is tested for residual live virusby assaying for growth on a suitable cell line.

The recombinant PPV can be used in immunogenic compositions andvaccines.

The immunogenic compositions and vaccines optionally can include one ormore veterinarily acceptable carriers, including liquid, semisolid, orsolid diluents, that serve as pharmaceutical vehicles, excipients, ormedia. As used herein, a “veterinarily-acceptable carrier” includes anyand all solvents, dispersion media, coatings, adjuvants, stabilizingagents, diluents, preservatives, antibacterial and antifungal agents,isotonic agents, adsorption delaying agents, and the like. Diluents caninclude water, saline, dextrose, ethanol, glycerol, and the like.Isotonic agents can include sodium chloride, dextrose, mannitol,sorbitol, and lactose, among others known to those skilled in the art.Stabilizers include albumin, among others known to the skilled artisan.Preservatives include merthiolate, among others known to the skilledartisan.

Adjuvants include, but are not limited to, the RIBI adjuvant system(Ribi Inc.), alum, aluminum hydroxide gel, oil-in water emulsions,water-in-oil emulsions such as, e.g., Freund's complete and incompleteadjuvants, Block co polymer (CytRx, Atlanta Ga.), SAF-M (Chiron,Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A, QS-21(Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (GalenicaPharmaceuticals, Inc., Birmingham, Ala.) or other saponin fractions,monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labileenterotoxin from E. coli (recombinant or otherwise), cholera toxin, ormuramyl dipeptide, among many others known to those skilled in the art.The amounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan. In one embodiment, the present invention contemplatesimmunogenic compositions and vaccines comprising from about 50 μg toabout 2000 μg of adjuvant. In another embodiment adjuvant is included inan amount from about 100 μg to about 1500 μg, or from about 250 μg toabout 1000 μg, or from about 350 μg to about 750 μg. In anotherembodiment, adjuvant is included in an amount of about 500 μg/2 ml doseof the immunogenic composition or vaccine.

The immunogenic compositions and vaccines can also include antibiotics.Such antibiotics include, but are not limited to, those from the classesof aminoglycosides, carbapenems, cephalosporins, glycopeptides,macrolides, penicillins, polypeptides, quinolones, sulfonamides, andtetracyclines. In one embodiment, the present invention contemplatesimmunogenic compositions and vaccines comprising from about 1 μg/ml toabout 60 μg/ml of antibiotic. In another embodiment, the immunogeniccompositions and vaccines comprise from about 5 μg/ml to about 55 μg/mlof antibiotic, or from about 10 μg/ml to about 50 μg/ml of antibiotic,or from about 15 μg/ml to about 45 μg/ml of antibiotic, or from about 20μg/ml to about 40 μg/ml of antibiotic, or from about 25 μg/ml to about35 μg/ml of antibiotic. In yet another embodiment, the immunogeniccompositions and vaccines comprise less than about 30 μg/ml ofantibiotic.

In addition to the recombinant PPV, immunogenic compositions andvaccines can include other antigens. Antigens can be in the form of aninactivated whole or partial preparation of the microorganism, or in theform of antigenic molecules obtained by genetic engineering techniquesor chemical synthesis. Other antigens appropriate for use in accordancewith the present invention include, but are not limited to, thosederived from pathogenic bacteria or pathogenic viruses.

In the case of dogs, the recombinant rabies immunogenic compositions andvaccines can also optionally contain a mixture with one or moreadditional canine antigens such as, for example, Ehrlichia canis, canineparvovirus (CPV), canine distemper, canine parainfluenza virus (CPI),canine adenovirus type II (CAV-2), canine adenovirus (CDV), caninecoronavirus (CCV), Leptospira icterohemorrhagiae (LI), Leptospiracanicola (LC), Leptospira grippotyphosa (LG), Leptospira pomona (LP),Borrelia burgdorferi, and the like. One combination of antigensencompasses isolates of canine Parvovirus, canine distemper, canineadenovirus and canine parainfluenza, with or without coronavirus andLeptospira (including the emerging serovars Leptospira grippotyphosa andLeptospira pomona).

In the case of cats, the recombinant rabies immunogenic compositions andvaccines can also optionally contain a mixture with one or moreadditional feline antigens such as, for example, feline calicivirus(FCV), Chlamydophila felis (C. felis, also previously and commonly knownas Chlamydia psittaci (FCP)), feline leukemia virus (FeLV), felinepanleukopenia virus (FPV), feline rhinotracheitis virus (FVR), felineimmunodeficiency virus (Fly), feline infectious peritonitis virus(FIPV), Bartonella henselae (e.g., cat scratch disease) and the like.

In the case of horses, the recombinant rabies immunogenic compositionsand vaccines can also optionally contain a mixture with one or moreadditional equine antigens such as, for example, Equine influenza virus,Equine herpesvirus 1 and 4, Equine arterivirus, West Nile virus, Equinerotavirus, Streptococcus equi, Tetanus toxoid, and the like.

Immunogenic compositions and vaccines described herein can beadministered to an animal to induce an effective immune response againstRV. Accordingly, described herein are methods of stimulating aneffective immune response against RV comprising administering to ananimal a therapeutically effective amount of an immunogenic compositionor vaccine comprising a recombinant parapoxvirus comprising heterologousDNA derived from a rabies virus. The method results in the induction ofanti-G protein serum antibodies.

Immunogenic compositions and vaccines described herein can beadministered to an animal to vaccinate the animal against rabiesdisease. The immunogenic compositions and vaccines can be administeredto the animal to prevent or treat rabies disease in the animal.Accordingly, described herein are methods of vaccinating an animalagainst rabies disease, and preventing or treating rabies disease,comprising administering to the animal a therapeutically effectiveamount of an immunogenic composition or vaccine comprising a recombinantparapoxvirus comprising heterologous DNA derived from a rabies virus.

Forms, Dosages, Routes of Administration

Immunogenic compositions and vaccines can be made in various formsdepending upon the route of administration. For example, the immunogeniccompositions and vaccines can be made in the form of sterile aqueoussolutions or dispersions suitable for injectable use, or made inlyophilized forms using freeze-drying techniques. Lyophilizedimmunogenic compositions and vaccines are typically maintained at about4° C., and can be reconstituted in a stabilizing solution, e.g., salineor and HEPES. Alternatively, immunogenic compositions and vaccines canbe preserved by freeze drying. Immunogenic compositions and vaccines canalso be made in the form of suspensions or emulsions.

Immunogenic compositions and vaccines include a therapeuticallyeffective amount of the above-described recombinant PPV. Purifiedviruses can be used directly in an immunogenic composition or vaccine,or can be further attenuated, or inactivated, Typically, an immunogeniccomposition or vaccine contains between about 1×10² and about 1×10¹²PFU, or between about 1×10³ and about 1×10¹¹ PFU, or between about 1×10⁴and about 1×10¹⁰ PFU, or between about 1×10⁵ and about 1×10⁹ PFU, orbetween about 1×10⁶ and about 1×10⁸ PFU. The precise amount of a virusin an immunogenic composition or vaccine effective to provide aprotective effect can be determined by a skilled artisan.

The immunogenic compositions and vaccines generally comprise aveterinarily acceptable carrier in a volume of between about 0.5 ml andabout 5 ml. In another embodiment the volume of the carrier is betweenabout 1 ml and about 4 ml, or between about 2 ml and about 3 ml. Inanother embodiment, the volume of the carrier is about 1 ml, or is about2 ml, or is about 3 ml, or is about 5 ml. Veterinarily acceptablecarriers suitable for use in immunogenic compositions and vaccines canbe any of those described herein.

Those skilled in the art can readily determine whether a virus needs tobe attenuated or inactivated before administration. In anotherembodiment, the recombinant PPV can be administered directly to ananimal without additional attenuation. The amount of a virus that istherapeutically effective can vary depending on any of several factorsincluding the condition of the animal and the degree of infection, andcan be determined by a skilled artisan.

In accordance with the methods of the present invention, a single dosecan be administered to animals, or, alternatively, two or moreinoculations can take place with intervals of from about two to aboutten weeks. Boosting regimens can be required and the dosage regimen canbe adjusted to provide optimal immunization. Those skilled in the artcan readily determine the optimal administration regimen.

Immunogenic compositions and vaccines can be administered directly intothe bloodstream, into muscle, or into an internal organ. Suitable meansfor parenteral administration include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular and subcutaneous. Suitabledevices for parenteral administration include needle (includingmicroneedle) injectors, needle-free injectors and infusion techniques.

Parenteral formulations are typically aqueous solutions which cancontain excipients such as salts, carbohydrates and buffering agents(preferably to a pH of from about 3 to about 9, or from about 4 to about8, or from about 5 to about 7.5, or from about 6 to about 7.5, or about7 to about 7.5), but, for some applications, they can be more suitablyformulated as a sterile non-aqueous solution or as a dried form to beused in conjunction with a suitable vehicle such as sterile,pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, forexample, by lyophilisation, can readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.

The recombinant parapoxviruses and immunogenic compositions and vaccinesdescribed herein can be used in the preparation of a medicament forvaccinating an animal against rabies disease.

The present invention provides methods of determining the origin of aparapoxvirus present in an animal.

Vaccination which utilizes a DIVA vaccine—one which is able todifferentiate infected from vaccinated animals—provides a means fordetermining the origin of a parapoxvirus present in an animal. Thisdifferentiation can be accomplished via any of various diagnosticmethods, including but not limited to ELISA, Western blotting and PCR.These and other methods are readily recognized and known to one ofordinary skill in the art.

The parapoxviruses described herein can be distinguished from wild-typestrains in both their genomic composition and proteins expressed. Suchdistinction allows for discrimination between vaccinated and infectedanimals. For example, a determination can be made as to whether ananimal testing positive for parapoxvirus in certain laboratory testscarries a wild-type parapoxvirus strain, or carries a recombinantlyproduced parapoxvirus previously obtained through vaccination.

A variety of assays can be employed for making the determination. Forexample, virus can be isolated from the animal testing positive forparapoxvirus, and nucleic acid-based assays can be used to determine thepresence of a parapoxvirus genome, indicative of prior vaccination. Thenucleic acid-based assays include Southern or Northern blot analysis,PCR, and sequencing. Alternatively, protein-based assays can beemployed. In protein-based assays, cells or tissues suspected of aninfection can be isolated from the animal testing positive forparapoxvirus. Cellular extracts can be made from such cells or tissuesand can be subjected to, e.g., Western Blot, using appropriateantibodies against viral proteins that can distinctively identify thepresence of either the recombinantly produced parapoxvirus previouslyinoculated, or wild-type parapoxvirus.

The extent and nature of the immune responses induced in the animal canbe assessed by using a variety of techniques. For example, sera can becollected from the inoculated animals and tested for the presence orabsence of antibodies specific for the parapoxvirus e.g. in aconventional ELISA. Detection of responding cytotoxic T-lymphocytes(CTLs) in lymphoid tissues can be achieved by assays such as T cellproliferation, as indicative of the induction of a cellular immuneresponse. The relevant techniques are well described in the art, e.g.,Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc.(1994).

The recombinant Parapoxvirus ovis D1701-V-RabG can be used in a DIVAassay. In one embodiment, it can be used in assays for the detection ofrabies N-genes or proteins to differentiate infected from vaccinatedanimals. In another embodiment, it can be used in assays for thedetection of rabies P-genes or proteins to differentiate infected fromvaccinated animals. In yet another embodiment, it can be used in assaysfor the detection of rabies L-genes or proteins to differentiateinfected from vaccinated animals. In still another embodiment, it can beused in assays for the detection of rabies M-genes or proteins todifferentiate infected from vaccinated animals.

The present invention is additionally described by the followingillustrative, non-limiting Examples.

EXAMPLES

A recombinant Parapoxvirus ovis, which comprises the gene encoding forthe G protein of RV, was generated. Expression of the G protein wasassessed, as was the immunostimulatory and protective properties of therecombinant virus against RV.

Example 1 Generation of Rabies G Protein Expressing Recombinant VirusD1701-V-RabG

A recombinantly-generated Parapoxvirus ovis, which contains the geneencoding for the G protein of RV, was generated. Expression of the Gprotein was assessed, as was the immunostimulatory and protectiveproperties of the recombinant virus against RV.

Construction of Transfer Plasmid.

For generation of the recombinant Parapoxvirus ovis D1701-V-RabG, theParapoxvirus ovis (PPVO) vector system (U.S. Pat. No. 6,365,393; Rzihaet al., 2000, J. Biotechnol., 83, 137-145; Fischer et al., 2003, J.Virol. 77, 9312-9323; Henkel et al., 2005, J. Viral. 79, 314-325) wasused. The Rabies virus G protein gene was chemically synthesized (BlueHeron Biotech; USA) and provided in a pUC plasmid. The complete G genewas isolated as a BamHI-EcoRI DNA fragment of 1.582 bp in size byagarose gel (0.8% w/v) electrophoresis and purified by Qiaex II gelextraction kit (Qiagen; Germany). The plasmid pdV-Rec1 (Fischer et al.,2003) was double-digested with BamHI and EcoRI and used for ligation(Fast ligation kit, Promega; Germany). After transformation of E. coliDH5αF′ (Invitrogen, Thermo Fisher Scientific; Germany) insert-positivecolonies were selected by EcoRI-BamHI restriction digestion of plasmidDNA. This resulted in transfer plasmid pdV-RabG (FIG. 1), which wereprepared by Qiagen Plasmid Maxi Kit (Qiagen; Germany) and used for DNAsequencing. To this end, primer ORF32N (SEQ ID NO: 1;5′-GCGCGCTGCGGGTGCGCTACCAATTCGCGC-3′) located upstream of the insertionsite and primer ORF31N (SEQ ID NO: 2;5′-GCATCCCGTTACCACCGGAGACCGACGCTCCC-3′) located downstream of theinsertion site in pdV-Rec1 was used as well as internal G-gene-specificprimers RabG-F (SEQ ID NO: 6; 5′-GGAGTCTCTCGTTATCATATCTC-3′) and RabG-R(SEQ ID NO: 7; 5′-CTAAACAAGGTGCTCAATTTCGT-3′), respectively. Thisallowed the determination of the complete sequence of the inserted Ggene (SEQ ID NO: 4).

Selection of Recombinants by Bluo-Gal Staining.

Vero cells (10⁶ cells) were infected with 0.1 MOI (multiplicity ofinfection) of the lacZ-expressing virus D1701-VrV, and 2 hr latertransfected with 2 μg of pdV-RabG plasmid DNA by nucleofection,according to the manufacturer's recommendation (Amaxa Nucleofector,Lonza; Germany). Virus lysates were harvested 3-4 days later, and usedfor titration on Vero cells in 6-well plates (Fisher Scientific;Germany). When plaques became visible, agarose-containing Bluo-Gal wasoverlaid as described (Fischer et al., 2003). Virus plaques having awhite appearance were picked, and the single plaque eluates (overnightat 4° C. in phosphate-buffered saline (PBS)) were used for simultaneousinfection of Vero cells (1×10⁵ cells) in single wells of a 48-wellplate.

Selection of Recombinants by Plaque-PCR.

Isolation of DNA from each virus plaque isolate was performed in amodification of Pasamontes et al. (J. Virol. Methods 35:137-141; 1991).Virus lysate (0.2 ml) was frozen (−70° C.) and thawed (37° C.) threetimes, and sonicated on ice 3 times for 20-30 sec (sonic waterbath).After phenol and chloroform extraction, 10 μg yeast tRNA or 3 μlGlycoBlue (Ambion; Germany) were added before ethanol precipitation. TheDNA pellet was washed twice with 70% (v/v) ethanol, and dissolved afterdrying in 14 μl aqua bidest.

For RabG-specific PCR, 4 μl of the DNA were mixed on ice with 1 μlprimer mix, consisting of 4.0 pmol RabG-F (SEQ ID NO: 6) and 4.0 pmolRabG-R (SEQ ID NO: 7) primers, and 5 μl ReddyMix 2×PCR (Abgene, ThermoFisher Scientific; Germany). PCR was performed in a Trio Thermoblock(Biometra; Germany) by incubating for 2 min at 98° C., followed by 40cycles for 1 min at 96° C., 30 sec at 60° C., 30 sec at 72° C., and afinal extension step for 2 min at 72° C. The PCR products were separatedin horizontal 1% (w/v) agarose-ethidiumbromide (0.3 microgram per ml)gels. Virus lysates from plaque isolates revealing the G gene-specificPCR fragment of 433 bp in size were diluted and further plaque-purifiedat least 3 times, using Bluo-Gal agarose overlay as described above.Finally, the DNA of recombinant virus plaque isolates positive for the Ggene were tested in a LacZ gene-specific PCR using 4 μl DNA, 3.95 pmolprimer lacZ-F (SEQ ID NO: 8; 5′-CGATACTGTCGTCGTCCCCTCAA-31, and 4.13pmol primer lacZ-R (SEQ ID NO: 9; 5′-CAACTCGCCGCACATCTGAACT-3′). Afteradding 5 μl AccuPrime SuperMix II (Invitrogen, Fisher Scientific;Germany), PCR was performed by heating for 2 min at 98° C., followed by40 cycles for 1 min at 96° C., 30 sec at 62° C., and 90 sec at 68° C.,with a final extension step for 2 min at 68° C. Separation of PCRproducts was performed as described above. The absence of the LacZgene-specific fragment of 508 bp in size demonstrated that thecorresponding recombinant virus plaque isolates were free of thelacZ-expressing parental virus D1701-VrV following three rounds ofplaque purification. After preparation of high titer virus stocks ofD1701-V-RabG, viral DNA was prepared as described below, and used fortesting in RabG-PCR and LacZ-PCR,

Immunohistochemical Staining of Recombinant Virus Plaques (IPMA).

Successful expression of inserted foreign gene was first assayed byIPMA, which involves immunohistochemical staining of recombinant virusplaques titrated on Vero cells in 24-well plates. After the appearanceof virus plaques, the medium was aspirated from each well, and the cellsdried by leaving the plate open for approximately 10 min in a laminarflow hood. Thereafter, the cells were fixed with ice-cold absolutemethanol at −20° C. for 15-20 min. After washing twice with ice-cold 1%(v/v) fetal calf serum (FCS) in PBS, the cells were blocked with PBScontaining 10% (v/v) FCS for 90 min at room temperature (RT). Afterincubation for 60 min at RT with the G protein-specific mouse monoclonalantibody G559, diluted 1:200 in 1% FCS in PBS (FLI-Tuebingen; Germany).After 3 washes with PBS-T (PBS containing 0.05% (v/v) Tween-20), aperoxidase-coupled anti-mouse secondary antibody (Jackson-ImmunoRes.,DIANOVA; Germany), diluted 1:2000, was added, and incubated for 60 minat RT. After thorough washing with PBS-T and PBS, substrate (Vector NovaRed, Axxora; Germany) was added as recommended by the manufacturer'sinstruction, until red-brown positive staining became visible. Asnegative controls, non-infected cells and cells infected with D-1701-VrVor D-1701-V were included. Virus plaques and infected cells were foundstrongly positive for Rabies virus G protein.

Example 2 Characterization of D1701-V-RabG Preparation of Virus Stocks.

To obtain high titer recombinant virus stocks, 10-20 T150 culture flasks(Greiner; Germany) were simultaneously infected with a MOI of 0.5. After3 days, approximately 80% cytopathogenic effect (CPE) was observed, andthe cells and supernatant of all flasks were harvested and collected forcentrifugation (2 hr at 13,000 rpm, 4° C.). The supernatant wascarefully removed, and the virus pellet was dissolved overnight at 4° C.in 1-2 ml PBS. The virus suspension was completely dispersed bysonification (Sonic cell disruptor, Branson; Germany) on ice using 3pulses (100 W) of 10 sec, (10 sec break between each pulse), followed bycentrifugation (500-700×g, 10 min, 4° C.) to remove cell debris. Thesupernatant was stored on ice, while the cell pellet was resuspended in1.0 ml PBS, and sonicated on ice (2 times for 20 sec, with a 10 secbreak in between, then once for 30 sec). After low speed centrifugation,the supernatant was combined with the first supernatant, divided intoaliquots, titrated, and stored at −70° C.

Characterization of Viral DNA.

Vero cells were infected with MOI 0.5, and harvested after 2-3 days(approx. 80% CPE) by trypsinisation and brief low speed centrifugationat 4° C. DNA was isolated using the Master Pure DNA Isolation Kit(Epicentre Biotechnology, Biozym Scientific; Germany), according to themanufacturer's protocol.

To verify insertion of the G gene in the correct locus, 2 μg DNA wererestriction enzyme-digested, separated in 0.8% (w/v) agarose gels, andtransferred to nylon membrane (GE Healthcare; Germany) for Southern blothybridization according to standard procedures. A Rabies G gene-specificprobe (the product of the Rabg-F/-R PCR) was gel-isolated, radioactivelylabeled (³²P-dCTP, MP Biomedicals; Germany) using RediPrime (GEHealthcare; Germany). This was then used for Southern blothybridization, carried out under conditions of 50° C. in 4×SSPE (1×=0.18M NaCl, 10 mM PP, 1 mM EDTA, pH 7.4) containing 0.5% (w/v) non-fat milkpowder, 1.0% (w/v) sodium.dodecylsulfate (SDS), and 0.5 mg/ml denaturedcalf thymus DNA (KT-DNA, Sigma; Germany). Following X-ray (Kodak X-Omat;Germany) exposure, the probe was removed from the filter by incubationin 0.4 N NaOH at 45° C. for 30-60 min, followed by brief incubation at100° C. in 0.1×SSC, 0.5% SDS, 0.2 M Tris-HCl (pH 7.4). For the secondhybridization, the HindIII fragment H of D1701-V containing the vegF-Elocus was used as described (Cottone et al., 1998. Virus Res. 56,53-67). Southern blot results confirmed the correct insertion of the Ggene into the genome of D1701-VrV.

Detection of G Gene-Specific RNA.

Vero cells were infected with a MOI of 3-5, and total RNA was isolatedat different times after infection (p.i.) using SurePrep Total RNAExtraction Kit (Fisher Scientific; Germany). Additionally, RNA wasextracted from cells infected in the presence of cytosine arabinoside(AraC; 0.04 mg/ml, Sigma; Germany) or cycloheximide (CHX, 0.1 mg/ml,Serva; Germany) to test for early expression of the inserted G gene. Asa control, RNA was isolated from non-infected cells. The RNAs wereseparated in a denaturing 1% agarose get containing formaldehyde, andtransferred to nylon membrane as described (Kroczek, R. A. & Siebert. E.Anal. Biochem, 184:90-95, 1990). The radioactively-labeled Rabies G PCR(RabG-F/-R) fragment was used as hybridization probe in UltraHybsolution (Ambion; Germany) at 42° C. The results clearly demonstratedimmediate early expression of the Rabies G gene, due to its regulationunder the control of the early vegF-E promoter of ORFV (Rziha et al.1999, J. Biotechnol., 73, 235-242)

Detection of G Protein by Immunofluorescence.

For immunofluorescence, Vero cells (1×10⁵ cells/ml) were infected in4-chamber slides (BD Falcon; Germany) with a MOI of 0.1 or 3.0. Atdifferent times p.i., the cells were washed with medium, and fixed with3.7% (v/v) methanol-free formaldehyde (Pierce, Thermo Fisher Scientific;Germany) for 15 min at 37° C. After 3 washes with PBS, the cells werepermeabilized by treatment with 0.2% (v/v) Triton X-100 for 5 min at 37°C. After PBS washing, the cells were blocked with 5% FCS in PBS for30-40 min at 37° C. For G protein detection, cells were incubated for 1hr at 37° C. with the mouse monoclonal antibody G559 (FLI; Tuebingen,Germany) diluted 1:1000 in PBS containing 1% FCS. After 5 washes in PBS,slides were incubated in the dark at 37° C. for 30 min with thesecondary anti-mouse Alexa-555 antibody, diluted 1:1000 in PBS(Molecular Probes; Germany).

Detection of cells at late times after infection with ORFV was carriedout by the use of ORFV-specific rabbit antiserum PAS2274, provided byDr. Rudiger Raue, (Pfizer, UK). The serum was diluted 1:100 in PBS with1% FCS, and secondary antibody, the anti-rabbit Alexa-488, was used at a1:1000 dilution.

Actin staining was performed with Phalloidin-647, according to theinstructions of the manufacturer (Biotium; Germany) followed by stainingof the nucleus with 0.04 μg/ml DAPI(4′,6-Diamidin-2′-phenylindoldihydrochlorid; Roche MolecularBiochemicals; Germany), for 20-30 min at RT in the dark. After thoroughwashing in PBS, the slides were embedded with Mowiol-DABCO, andfluorescence imaging was performed with a Zeiss ApoTome, usingAxiovision software.

The results clearly demonstrated strong expression of the Rabies Gprotein early (4 hr p.i.) as well as late (24 h p.i.) after infectionwith D1701-V-RabG. Cells infected late could be additionally identifiedby specific staining with antiserum PAS2274. Moreover, the fluorescencestaining proved evidence of surface expression of the G protein.Specificity of staining was tested via the use of non-infected cells.

Detection of G Protein by Western Blotting

Vero cells (3×10⁵ cells) were simultaneously infected with a MOI of 1.0,and incubated at 37° C. in a 5% CO₂ atmosphere. At different times p.i.,the cells plus supernatant were harvested, centrifuged (8,900×g, 10 min,4° C.), and the cell sediment was washed 3 times with 1.0 ml PBS andresuspended in 0.15 ml PBS containing 1% (v/v) Triton X-100. After 30min on ice, the lysate was centrifuged for 15 min at 15.000×g, 4° C.,and the supernatant saved for SDS-PAGE (Polyacrylamide gelelectrophoresis). To this end, 3 parts of lysate were mixed with onepart 4× DualColor protein loading buffer (Fermentas; Germany), boiledfor 5 min, sonicated, and approx. 10 μg protein was separated bySDS-PAGE using 8% (w/v) ProSieve50 gel with Tris-Tricine-SDS runningbuffer as recommended (FMC Bioproducts, Biozym; Germany). The PrestainedProtein Ladder (Fermentas; Germany) was used as molecular weightmarkers. After electrophoresis, the proteins were transferred to a PVDFmembrane according to the instruction of the manufacturer (Pierce,Thermo Fisher Scientific; Germany). After membrane blocking in 3×Rotiblock (Roth; Germany) for 3 hours at room temperature, a rabbitanti-peptide antiserum specific for the C-terminus of the Rabies virus Gprotein (kindly provided by Dr. K.-K. Conzelmann, Max-von-PettenkoferInstitute; Munich, Germany) was used 1:10,000, diluted in 1× RotiBlock.After overnight incubation at 4° C., the membrane was thoroughly washed5 times in TBS-T (Tris-buffered saline with 0.05% v/v Tween-20), andincubated with a peroxidase-coupled anti-rabbit antibody (1:20,000;Jackson-ImmunoRes., Dianova; Germany) for 1 hr at RT. After TBS-Twashing, ECL substrate was used as recommended (Immobilon Western,Millipore; Germany). The reacted proteins were detected by the use ofchemiluminescence X-ray film (CL-XPosure, Pierce, Thermo FisherScientific; Germany).

Expression of the Rabies virus G protein of the expected molecularweight (58-60 kDa) was confirmed at the different times after infection.

Example 3 Induction of Specific Immune Response after Immunization ofMice with D1701-V-RabG Dose Dependent Induction of Anti-G SerumAntibodies.

The G protein can be regarded as the most important antigen for aprotective immune response, and the extent of the inducedvirus-neutralizing serum antibodies (SNT) can be correlated withprotection against Rabies virus challenge infection. According to theOIE (World Organization of Animal Health) and WHO (World HealthOrganization), the presence of antibody titers exceeding 0.5 to 1.0IU/ml (International unit) of SNT are regarded as protective. Therefore,the induction of G protein-specific SNT antibodies was determined duringday 1 to day 14 following prime immunization of mice with D1701-V-RabG.

C57/BL6 mice of 6-8 weeks of age (n 6 or 7 per group), bred at the FLI(Friedrich-Loeffler-Institute, Federal Research Institute of AnimalHealth, Institute of Immunology; Tuebingen, Germany), wereintramuscularly immunized with 0.1 ml of the indicated PFU(plaque-forming units) of D1701-V-RabG (0.05 ml for the thigh of eachhind leg). Individual serum samples were taken daily, and used fordetermination of SNT in a rapid fluorescence focus inhibition test(RFFIT) as described. (OIE Manual of Diagnostic Tests and Vaccines forTerrestrial Animals. 2007, Part 2, Section 2.2, Chapter 2.2.5 Rabies,which is also found at the following Internet website:www.oie.int/fr/normes/mmanual/A_(—)00044.htm; Cox, J. H. and L. G.Schneider, 1976, J. Clin. Microbial. 3, 96-101.) In brief, serial 5-foldserum dilutions in RPMI medium were prepared, and 0.05 ml of eachdilution was mixed (in duplicate) in the wells of a 96-well plate,together with 0.05 ml Rabies virus, strain CVS 11 (lot no. 47, 1.7×10⁵PFU/ml). After 90 min incubation at 37° C. and 5% CO₂, 0.1 ml of a BHK21cell suspension (1×10⁶ cells/ml) was added into each well, mixed, andincubated 24 hours at 37° C. in 5% CO₂. After washing the wells of theculture plate with PBS and pre-chilled 80% (v/v) acetone, the cells werefixed in fresh 80% (v/v) acetone for additional 30 min at 4° C. Afterremoval of the acetone and air drying, 0.05 ml of FITC anti-rabiesmonoclonal globulin (Centocor; USA) was added for 30 min at 37° C. tostain the Rabies virus-infected cells. After washing twice with PBS andonce with aqua bidest, the virus infection was monitored by fluorescencemicroscopy. Serum dilutions that reduced the number of infected cells to50% were read as positive. The titers of the sera were expressed asIU/ml, and were compared to positive WHO reference serum.

FIG. 2 demonstrates the development of SNT after a single intramuscularimmunization of mice with the indicated PFU of D1701-V-RabG recombinantvirus. Sera were tested on the indicated days (d) after immunization byFFIT. As can be seen in FIG. 2(D), even low dose (10⁴ PFU) immunizationresulted, by day 8, in a mean SNT of 5.5 IU/ml, increasing until day 14after immunization to approximately 13 IU/ml. Using 10⁶ or 10⁷ PFU forimmunization (FIGS. 2A and 2B), by one week later, a mean SNT ofapproximately 10-20 IU/ml was induced, respectively, which increased upto approximately 50 IU/ml 14 days after priming. Using 10⁷ PFU ofD1701-V-RabG, by day 4 after immunization, protective serum antibodytiters (0.6-3.0 IU/ml) were detected, which was not the case using thetested lower immunization doses (FIG. 2).

Another mouse experiment (data not shown) demonstrated that a boosterimmunization using either 10⁶ or 10⁷ PFU of the recombinant virus led toonly a marginal increase in the SNT 14 days after prime immunization(with 10⁶ or 10⁷ PFU).

Collectively, these results demonstrate the successful induction ofprotective SNT antibody titers during the first week after immunizationof mice with various doses of D1701-V-RabG.

Example 4 Protective Immune Response in Mice Mediated by D1701-V-RabG

An evaluation of the protective capacity of D1701-V-RabG was performedby challenge infection of different immunized mice (C57/BL6) accordingto the recommendations of the DIE (Manual of Diagnostic Tests andVaccines for Terrestrial Animals, 2007. Part 2, Section 2.2, Chapter2.2.5 Rabies. Also found atwww.oie.int/fr/normes/mmanual/A_(—)00044.htm.). At an age of 6-8 weeks,mice (n=11 or 12 per group) were immunized with the indicated PFU ofD1701-V-RabG as described above. Non-immunized control mice (n=15) wereinjected with PBS. Three weeks after immunization, all animals wereintracranially challenged with virulent rabies virus strain CVS (0.03 mlcontaining 4.8×10⁵ PFU). The animals were observed daily until 21 daysafter challenge infection. Moribund animals suffering from severeneuronal symptoms were euthanized.

As shown in FIG. 3, a single intramuscular immunization with 10⁷ PFU ofD1701-V-RabG completely protected the mice (11/11) against a high doseof intracranially applied challenge virus. One immunized animal fromthat group died on day 12, but this was not due to the challenge Rabiesvirus infection. Therefore, that animal was excluded from the dataanalysis. Application of one dose containing a 10-fold lesser amount(10⁶ PFU) of D1701-V-RabG still achieved 73% protection (8/11survivors). Further decrease of the immunization dose reduced theprotective rate to 58% (10⁵ PFU; 7/12), or to 27% (10⁴ PFU; 3/11),whereas all control immunized mice (n=15) died during day 6 and 9 afterchallenge infection (FIG. 3).

Example 5 Role of T-Cells for Protective Immunity

The following experiments investigated the contribution of T cells(CD4-, CD8-, or CD4/8-positive cells) for the induction of protectiveimmunity by the recombinant D1701-V-RabG in mice. As indicated in FIG.5A, just prior to and following the Day-0 immunization with 10⁷ PFU ofD1701-V-RabG (i.e., on Days −1, 0, +1 and +5), antisera specific forCD4- or CD8-T-cells were administered intraperitoneally to groups ofmice (number (n) of animals shown in the Figure) to deplete each T-cellpopulation in vivo.

Thereafter, on Day 15, all animals were challenged intracerebrally witha dose of 500 LD₅₀ of virulent RV strain CVS.

As shown in FIG. 5A, animals depleted for CD4-positive T-cells generallywere not protected, as seen by the similar response to the non-immunizedcontrol animals.

As seen in FIG. 5B, groups of animals were immunized on Day 0 withD1701-V-RabG, and then in vivo depleted of CD4- and/or CD8-T-cells justprior to and following the Day-15 challenge infection with the virulentRV strain CVS, i.e., on Days 13 (2 days before challenge infection), 15,19, and 23. The results demonstrate that after successful priming of theanti-Rabies virus immune response by D1701-V-RabG, the depletion ofT-cells 14 days later did not adversely affect protection, in that 75-90percent of animals that were in vivo depleted of T-cell populationssurvived the challenge.

Conclusively, the results show that CD4-positive T-cells (most probablyT-helper cells necessary for anti-G antibody production) are the majordeterminant of the protective immunity induced by the recombinantD1701-V-RabG.

Example 6 Post-Exposure Vaccination

The efficacy of the recombinant D1701-V-RabG for therapeutic vaccinationwas tested in mice. To this end, groups of animals were vaccinatedintramuscularly (i.m.) with 10⁷ PFU of D1701-V-RabG on Days 0, 1, and 4.The mice were challenged with 1×10⁶ PFU of virulent RV strain CVS on Day0. As shown in FIG. 6, all mice in the immunized group except one wereprotected against rabies.

Additional experiments were performed in mice to test variouspost-exposure immunization regimens to the virulent RV strain CVS. InFIG. 7, gray squares indicate the days on which mice were vaccinatedwith D1701-V-RabG. Results (survivors) are summarized in the Figure.

The results demonstrate the capability of D1701-V-RabG for therapeuticvaccination of mice. As seen in FIG. 7, a dual vaccination, given on theday of challenge and the following day, mediated 80% protection, and atleast 60% of animals were protected by 4 daily immunizations beginning 3days after the peripheral RV challenge infection.

Example 7 Immune Response Induced by Different Routes of Immunization

Mice were immunized with 1×10⁶ PFU of D1701-V-RabG by the following ofapplication: intravaginally (i.vag.), by scarification,intraperitoneally (i.p.), intradermally (i.d.), intransally (i.n.),subcutaneously (s.c.), intramuscularly (i.m.), and intraveneously(i.v.). The induced serum antibody response (determined as serumneutralization antibodies, or SNT) 6 days (gray bars) and 13 days (blackbars) after immunization is depicted in FIG. 8. Fourteen days afterimmunization the animals were intracerebrally infected with 100 LD₅₀ ofvirulent RV strain CVS, and the percent survivors was calculated.

The results show that the highest SNT titers (given as IU per ml) wereinduced by i.v., i.p., and i.m. vaccination, which also resulted in thebest protection rates of 86%, 100%, and 78%, respectively.

1. A recombinant parapoxvirus comprising a parapoxvirus and heterologousDNA derived from a rabies virus.
 2. The recombinant parapoxvirus ofclaim 1, wherein the parapoxvirus is a Parapoxvirus ovis virus (ORFV).3. The recombinant parapoxvirus of claim 2, wherein the parapoxvirus isParapoxvirus ovis strain D1701.
 4. The recombinant parapoxvirus of claim1, wherein the heterologous DNA comprises the gene encoding the Gprotein of the rabies virus, or fragments thereof.
 5. The recombinantparapoxvirus of claim 1, wherein the heterologous DNA comprises SEQ IDNO: 4, or a sequence having at least about 98% identity to SEQ ID NO: 4.6. The recombinant parapoxvirus of claim 1, wherein the heterologous DNAis inserted within the HindIII fragment H/H of Parapoxvirus ovis strainD1701.
 7. The recombinant parapoxvirus of claim 6, wherein theheterologous DNA is inserted within the VEGF coding sequence or adjacentnon-coding sequences within the HindIII fragment HUH of Parapoxvirusovis strain D1701.
 8. The parapoxvirus of claim 1, wherein theparapoxvirus is Parapoxvirus ovis D1701-V-RabG.
 9. A method of preparingthe recombinant parapoxvirus of claim 1 comprising insertingheterologous DNA into the genome of the parapoxvirus.
 10. The method ofclaim 9, wherein the parapoxvirus is Parapoxvirus ovis.
 11. The methodof claim 10, wherein the parapoxvirus is Parapoxvirus ovis strain D1701.
 12. The method of claim 9, wherein the heterologous DNA comprisesthe gene encoding the G protein of the rabies virus, or fragmentsthereof.
 13. The method of claim 9, wherein the heterologous DNAcomprises SEQ ID NO: 4, Or a sequence having at least about 98% identityto SEQ ID NO:
 4. 14. The method of claim 9, wherein the recombinantparapoxvirus is Parapoxvirus ovis D1701-V-RabG.
 15. An immunogeniccomposition comprising the recombinant parapoxvirus of any of claims 1to 8 and a carrier.
 16. A method of preparing the immunogeniccomposition of claim 15, comprising combining the recombinantparapoxvirus with a carrier.
 17. A method of inducing an immune responseagainst rabies virus in an animal comprising administering to saidanimal an immunologically effective amount of an immunogenic compositionof claim
 15. 18. The method of claim 17, wherein the immune response isthe induction of anti-G protein serum antibodies.
 19. The method ofclaim 17, wherein an anti-G protein-specific protective immune responseis induced.
 20. The method of claim 19, wherein the induction results inantibody titers exceeding 0.5 International Units per ml.
 21. Use of therecombinant parapoxvirus of any one of claims 1 to 8 in the preparationof a medicament for inducing an immune response against rabies virus inan animal.
 22. A use of the recombinant parapoxvirus of any one ofclaims 1 to 8 in an assay for the differentiation of infected fromvaccinated animals.